Putting 2 plasmids in 1 e. coli cell - (May/31/2005 )

Hello!

I am co-expressing 2 proteins and I have them in 2 seperate vectors (pET15 and pET28). One vector codes for ampicilin resistance, the other for kanamycin. My question is, can I mix a solution of the 2 plasmids and comp them into a cell at once? I figure if I spread it on an amp/kan plate and get colonies I will have comped both.

The other option I am considering is doing 1 plasmid at a time. For example, put the pET15 in, make a culture of that competant, and then put in the pET28. I'm just not sure how many plasmids one e.coli cell can take up?

If anyone has experienced this problem, any info would be helpful.

-JediBill-

Yes, cotransformation is possible. Just mix the two plasmids, do the transformation (maybe use more plasmid than usual), and plate the cells on plates with two antibiotics.

-labrat-

Excellent - I will try that tonight!!

Thanks so much!

-JediBill-

Well, both vectors contain the same pBR322-ori, therefore they belong to thesame incompatibility group and even with selection I would expect not optimalexpression of your product. Especially at later stages of the growth curveselection pressure is relieved because both antibiotics are inactivated by thebeta-lactamase and the phosphotransferase.I suggest using a pBR322- and a pACYC-derivative (pSC101-ori), both orisare compatible.

Regards,Hennry

-hennry-

HelloI am planning to cotransform E.coli cells with the vectors.I have already cloned one of my inserts into pGEX-4T2 vector.For the other insert which vector should I choose for efficient cotransformation, selection and expression?thank youernie...

QUOTE (hennry @ Jun 1 2005, 01:37 PM)

Well, both vectors contain the same pBR322-ori, therefore they belong to thesame incompatibility group and even with selection I would expect not optimalexpression of your product. Especially at later stages of the growth curveselection pressure is relieved because both antibiotics are inactivated by thebeta-lactamase and the phosphotransferase.I suggest using a pBR322- and a pACYC-derivative (pSC101-ori), both orisare compatible.